Cytoprotective effects and antioxidant activities of acteoside and various extracts of Clerodendrum cyrtophyllum Turcz leaves against t-BHP induced oxidative damage

This study evaluates the antioxidant potential and cytoprotective effects of ethanolic crude extract from Clerodendrum cyrtophyllum leaves (ECE) and five derived fractions (namely, petroleum ether fraction (PEF), dichloromethane fraction (DMF), ethyl acetate fraction (EAF), n-butyl alcohol fraction (BAF) and the remaining fraction (RF)), as well as acteoside (Ac, a major phenolic component in EAF) on oxidative damage caused by tert-butyl hydroperoxide (t-BHP) in HepG2 cells. MTT assay results showed that ECE, EAF, BAF, RF and Ac increased the viability of t-BHP-damaged cells in a dose-dependent manner, while EAF significantly promoted cell viability. EAF, BAF, RF, or Ac reduced the levels of lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA), and reactive oxygen species (ROS). Additionally, glutathione (GSH) levels and the activities of superoxide dismutase (SOD) and catalase (CAT) increased. Western blot analysis further indicated that EAF, BAF, RF, or Ac up-regulated pro-caspase-3 and reduced cleaved caspase-3 during t-BHP-induced oxidative stress. Flow cytometry analysis and fluorescence micrographs showed that Ac could inhibit apoptosis.


Effect of extracts on the viability of HepG2 cells under oxidative stress. As shown in
, treatment with 700 μmol/L t-BHP for 3 h caused the viability of HepG2 cells to decrease to about 50.0%, compared with the control group; hence, this concentration was used to simulate oxidative damage in HepG2 cells in subsequent experiments.
Considering the extract itself may be toxic to cells, we determined the effects of all extracts (ECE, PEF, DMF, EAF, BAF, and RF) on the viability of HepG2 cells without the presence of t-BHP. The results show that the viability of extract-treated HepG2 cells was not significantly different from that of the control group (p > 0.05). The six extracts (at 10, 20, and 30 μg/mL) neither promoted nor decreased the viability of HepG2 cells (Fig. 2B). This finding indicates that all extracts are not cytotoxic and do not interfere with the cell viability assay of t-BHP.
We further evaluated the cytoprotective effects of the extracts against t-BHP-induced oxidative damage. As shown in Fig. 2C, treatment with 700 μmol/L t-BHP caused the survival rate of HepG2 cells to reduce by about 50%, compared to that of the control group. Additionally, the survival rate of HepG2 cells pretreated with ECE increased with increasing concentration of ECE. The survival rate of HepG2 cells treated with crude material at 20 and 30 μg/mL was significantly higher than that of t-BHP-treated group (p < 0.05). Furthermore, the survival rate of HepG2 cells pretreated with EAF, BAF, and RF increased with increasing sample concentration; however, both PEF and DMF at all concentrations had no significant effect on survival rate. At the same concentration of 30 μg/mL, the survival rate of HepG2 cells treated with EAF was significantly higher than that Effect of extracts on the accumulation of intracellular ROS. As illustrated in Fig. 3A, t-BHP clearly increased cellular ROS levels (by ~ twofold), but cells pretreated with ECE, EAF, BAF, and RF exhibited reduced ROS generation of 13.8%, 25.1%, 24.8% and 13.2% (p < 0.05), respectively, compared to t-BHP-stressed cells. In contrast, the reduction in ROS levels of PEF-and DMF-treated cells was not statistically significant. The above results demonstrate that ECE, EAF, BAF, and RF act as scavengers of the ROS generated by t-BHP in HepG2 cells.
Effect of extracts on LDH release and MDA production. In order to evaluate the possible mechanism involved in the protective effect of C. cyrtophyllum extracts on cytotoxicity, LDH release, and MDA production were evaluated. Figure 3B,C shows that t-BHP-induced HepG2 cells pretreated with 100 μL of the 30 μg/mL C. cyrtophyllum extracts for 6 h exhibited LDH release and MDA level decreased. As compared with t-BHPinduced HepG2 cells (p < 0.05), indicating that ECE, EAF, BAF, and RF markedly attenuated oxidative stress of lipid peroxidation. Fig. 3D, 700 μmol/L t-BHP significantly increased the cytoplasmic GSH level of the cells (p < 0.05); however, the GSH levels sharply decreased after the cells were treated with 100 μL of 30 μg/mL ECE, EAF, BAF, or RF for 6 h. In contrast, the GSH contents in the cells treated with PEF and DMF were not significantly different from those in the cells treated with t-BHP. Furthermore, to characterize the antioxidant enzymes of HepG2 cells exposed to t-BHP, the activities of CAT and SOD were determined. As illustrated in Fig. 3E,F, pretreatment with ECE, PEF, DMF, EAF, BAF, or RF at 30 μg/mL significantly increased the activities of SOD and CAT (p < 0.05). EAF showed the highest SOD and CAT activities compared to that of cells treated with t-BHP. However, cells pretreated with PEF did not affect SOD and CAT activities.

Effect of extracts on GSH level and SOD and CAT activities. As shown in
Effects of extracts on apoptosis protein caspase-3. Expression of caspase-3 in HepG2 cells was evaluated in our study, and the results are shown in Fig. 4A,B. Treatment with 700 μmol/L t-BHP significantly reduced the level of pro-caspase-3 (43.8%) and induced caspase-3 activation to 130.8%. As expected, pretreatment with EAF, BAF, and RF opposed these effects, and the pro-caspase-3 level significantly increased by 65.7%,  Effect of Ac on the viability of HepG2 cells under oxidative stress. The cytotoxic effect of acteoside (Ac) on HepG2 cells is shown in Fig. 2D. After 6 h treatment with Ac at concentrations between 1 and 600 μmol/L, there was no significant toxicity in cell viability, compared to the control group. However, cell viability dropped slightly at 1000 μmol/L Ac. We further evaluated the cytoprotective effects of Ac against t-BHPinduced oxidative damage. As shown in Fig. 2E, pre-incubation of HepG2 cells with Ac (1-400 μmol/L) for 6 h reduced oxidative damage induced by t-BHP, increasing cell viability in a dose-dependent manner. This result was significantly different from that of the damage control. At the same concentration of 200 μmol/L, Ac and BHT promoted higher cell viability than VE against t-BHP-induced oxidative stress. These data demonstrate that Ac exhibits a remarkable cytoprotective capacity.

Effect of Ac on GSH level and SOD and CAT activities.
To further determine the likely transcriptional mechanism underlying the protective effects of Ac, GSH level and the activities of intracellular antioxidant enzymes SOD and CAT were examined. As shown in Fig. 5D-F, exposure of HepG2 cells to 700 μmol/L t-BHP dramatically decreased the GSH level, as well as SOD and CAT activities. Pre-incubation of cells with 10-200 μmol/L Ac for 6 h attenuated these effects in a dose-dependent manner. At 200 μmol/L, Ac exhibited a greater increase in GSH and SOD and CAT activities than BHT and VE.

Effects of Ac on apoptosis.
To investigate the effect of Ac against the apoptosis response of HepG2 cells induced by oxidative stress, the expression of the apoptosis-related protein, caspase-3, was determined by western blot. As illustrated in Fig. 6A,B, treatment of HepG2 cells with Ac, followed by t-BHP, could increase the amount of anti-apoptotic pro-caspase-3 and reduce the amount of pro-apoptotic cleaved-caspase-3 in a dosedependent manner.

Discussion
Oxidative stress is an essential factor that causes many chronic diseases, and it is closely related to the accumulation of ROS. For example, the liver disease usually results in excess ROS production in the body 18 . Proper use of antioxidants is of great significance in preventing oxidative-related disorders and, in some cases, helps in their treatment 19 . Phytochemical or antioxidant therapies are considered promising strategies to protect cells from oxidative damage, and plant-derived natural products are important sources of antioxidants 20 . Various studies have screened different plant extracts for their antioxidant potential 15,21,22 . Particularly, polyphenols have proven their efficacy as antioxidants and have been adopted in the prevention and/or treatment of various human diseases 23 .
In this study, we demonstrated the antioxidant potential and cytoprotective effects of ethanolic crude extract from C. cyrtophyllum leaves (ECE) and five derived fractions, PEF, EAF, BAF, and RF, as well as polyphenolic Ac (Fig. 1). The results furthermore confirmed the antioxidant effects of C. cyrtophyllum extracts 9 .
High levels of dietary compounds can be toxic and mutagenic in cell culture systems 24 . None of the extracts (ECE, PEF, DMF, EAF, BAF, and RF) at 30 μg/mL were significantly toxic to HepG2 cells viability compared to the control group ( Fig. 2A). In accordance with a previous report on the physiological effects of Ac 25 , our study found Ac was not toxic in cellular physiological contexts. However, Ac did increase the oxidative load, affect the activity of proteostatic modules, and suppress matrix metalloproteinases in tumor cell lines. t-BHP is often used   17,26 . In the present study, we observed that the treatment of cells with t-BHP caused an increase of intracellular ROS content and resulted in decreased viability of HepG2 cells (Fig. 2B), but incubation with ECE or different polar extracts could mitigate apoptosis (Fig. 2C). Oxidative damage in hepatocytes was induced by the treatment of t-BHP, an organic hydroperoxide that can be metabolized to free radicals, making it a pivotal contributor to intracellular oxidative stress 22 . The antioxidant capacity can be evaluated by measuring the ability to scavenge ROS in t-BHP oxidative damage cell models 3 . Breakdown of the endogenous antioxidant defense mechanism can be reflected by the amount of LDH released, MDA levels, GSH levels, and SOD and CAT activities 27,28 . According to our results, after HepG2 cells were exposed to ECE, the amount of LDH released and the MDA level decreased. Conversely, GSH levels, SOD activity, and CAT activity significantly increased, indicating that ECE can effectively remove intracellular ROS. Similar results were also observed in EAF, BAF, and RF; and EAF was the most effective among all extracts (Fig. 3A-F). It is possible that EAF contains high concentrations of phenols (the major antioxidant constituents of C. cyrtophyllum), which have antioxidant activity and can be dissolved in a polar solvent 8 .
Oxidative stress plays a role as a common mediator to provoke cytosolic reaction cascades involving caspases and regulatory factors, such as caspase-3. Caspase-3 is a key terminal cleavage enzyme in the process of apoptosis, eventually leading to caspase-dependent and caspase-independent cell death 29,30 . In the present study, HepG2 cells were treated with 30 μg/mL C. cyrtophyllum extracts for 6 h before being exposed to t-BHP (700 μmol/L) for 3 h. The expression of caspase-3 was using measured western blot. ECE, especially for EAF, BAF, and RF, significantly up-regulated the expression of pro-caspase-3 and down-regulated the expression of cleaved caspase-3 (Fig. 4A,B). It is believed that ECE may interact with pro-caspase-3 to prevent cleavage by caspase-3.
To further investigate the underlying endogenous antioxidant defense mechanisms of C. cyrtophyllum, Ac was found to be a significant component (0.803 g, 0.54%) isolated from EAF. Reportedly, Ac exerts protective effects against 1-methyl-4-phenylpyridinium ion (MPP + )-induced oxidative stress in pheochromocytoma (PC12) and SH-SY5Y neuronal cells 31,32 . It is capable of reducing the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and MDA in the liver of mice injured by Bacillus Calmette-Guerin (BCG)/LPS 33 . Wang and coworkers 34 demonstrated the protective effects of Ac on amyloid β-protein-induced human neuroblastoma SH-SY5Y cell injury by inhibiting caspase-3 activation. In this study, Ac showed no apparent toxic or promotional effects in physiological cellular contexts at 1-600 μmol/L and significantly increased the viability of t-BHP-induced HepG2 cells (Fig. 2D,E). We found that 700 μmol/L t-BHP elevated intracellular ROS, leading to a roughly two-fold increase in fluorescence intensity. Pretreatment with Ac (10-200 μmol/L) for 6 h greatly reduced the production of ROS (Fig. 5A). Additionally, it reduced the amount of LDH released (Fig. 5B), as well as MDA levels (Fig. 5C), but it increased GSH levels (Fig. 5D), SOD activity (Fig. 5E), and CAT activity (Fig. 5F), HepG2 cells were treated with 30 μg/mL samples for 6 h before being exposed to TBHP (700 μmol/L) for 3 h. ROS level, LDH activity, MDA level, GSH level, SOD activity, and CAT activity in HepG2 cells were detected through the kit. Different letters above bars indicate significant difference (by ANOVA, p < 0.05). Results are expressed as mean ± SD (n ≥ 3). Ac, acteoside; BHT, butylated hydroxytoluene; VE, vitamin E; + , treated; −, untreated. www.nature.com/scientificreports/ compared to the positive control BHT and VE. Thus, it is evident that Ac can reduce oxidative stress damage in cells by inhibiting the production of ROS, reducing the production of peroxidation products, and enhancing the activity of antioxidant enzymes. Furthermore, Ac could up-regulate pro-caspase-3 while down-regulating cleaved caspase-3, which is evidenced by the ability of Ac to suppress TBHP-induced HepG2 cells apoptosis (Fig. 6A,B). Flow cytometry data and fluorescence micrographs provide reliable and clearly distinguished results between subpopulation of apoptotic cells and were closely intercorrelated 35 . We found that Ac enhanced the viability of HepG2 cells under oxidative stress and reduced the number of early and late apoptotic cells. (Fig. 7A-C). These results are in good agreement with the effects of Ac in preventing apoptosis.

Conclusions
In conclusion, we demonstrated that ethanol crude extracts from C. cyrtophyllum leaves could reduce the oxidative damage caused by t-BHP to HepG2 cells. Among all derived fractions, ethyl acetate fraction had the highest protective effect, followed by n-butanol fraction and the water fraction. The extracts could slow down the apoptosis of mitochondria by inhibiting the production of ROS. Moreover, enhanced levels of antioxidants and elevated activities of antioxidant enzymes in cells were also measured. Ac is naturally found in C. cyrtophyllum leaves, and it inhibits the production of ROS and prevents apoptosis by inhibiting the activation of caspase-3. Ac has shown an excellent cytoprotective effects and antioxidant activities against t-BHP induced oxidative damage, equal to or better than synthetic antioxidants BHT and natural antioxidants VE. Ac may be the main active component of C. cyrtophyllum, with desirable antioxidant activity and the potential to develop into plant originated antioxidant. The location we collect our plant materials is a national geological garden and the author was not obliged to have any permissions. This work did not involve endangered or protected species, the species C. cyrtophyllum is a common plant growing nearby the curbside. The study's authors promise the use of plants in the present study complies with international, national and/or institutional guidelines. A voucher specimen of the plant (P-DQ001) was deposited in the herbarium of Laboratory of Tropical Pharmaceutical Biomolecules, School of Chemical Engineering and Technology, Hainan University.
Preparation of extracts and acteoside. The ethanol extract of C. cyrtophyllum leaves (ECE) was first extracted with 49% ethanol for 85 min, using ultrasonic-assisted extraction. ECE was then subjected to the following extractions: ECE was extracted with pure petroleum ether to obtain petroleum ether fraction (PEF); ECE was extracted with pure dichloromethane to obtain dichloromethane fraction (DMF); ECE was extracted with pure ethyl acetate to obtain ethyl acetate fraction (EAF); ECE was extracted with n-butanol to obtain n-butanol fraction (BAF). The remaining ECE was a residual aqueous fraction (RF). All extracts were frozen, dehydrated, and stored at − 20 °C. Acteoside was isolated from the EAF fraction using column chromatography. All extracts,  Cell viability assay. Cell viability was determined by MTT assay. HepG2 cells were seeded in a 96-well plate at a density of 1 × 10 5 cells/well and incubated at 37 °C for 24 h. The cells were then treated with the above extracts for 6 h. Subsequently, 0.5 mg/mL MTT reagent dissolved in MEM was added to each well, and the plate was further incubated at 37 °C for 3 h. After the culture medium was discarded, the cells were washed once with PBS (pH 7.2), and DMSO was added to solubilize the formazan product. Absorbance was then measured at 570 nm using a micro-plate reader (KeHua ST-360, Shanghai, China). Western blot analysis. Western blot analysis was performed following the method described by Liu et al. 23 with slight modification. HepG2 cells were cultured in 6-well plates and treated with samples and t-BHP, as described above. After treatment, the cells were washed three times with PBS and were treated with RIPA lysate in a refrigerator (4 °C) for 2 h. The samples were then centrifuged at 12,000 rpm for 10 min. The lysate was collected, and the concentration of protein in the supernatant was determined using BCA assay. Proteins were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% polyacrylamide gel. The protein bands were transferred to a polyvinylidene fluoride (PVDF) membrane. After incubating with 5% skim milk in TBST buffer for 2 h at room temperature on a shaker, the membrane was incubated with primary antibody for 2 h at room temperature. Subsequently, the membrane was washed three times with TBST buffer for 5 min each and was then incubated with a secondary antibody for 1 h at room temperature. After the membrane was washed three times with TBST buffer, the bands on the membrane were visualized using an ECL system; the band density was optically scanned using ImageJ software.

Measurement of intracellular
Annexin V-FITC/PI double staining. HpeG2 cells were seeded in 6-well plates and incubated with or without acteoside, BHT, and VE at various doses for 6 h. The cells were then treated with 700 μmol/L t-BHP for 3 h. After washing with PBS, the cells were dissolved in 500 μL of 1 × binding buffer and stained with 5 μL Annexin-V and 10 μL PI (Multisciences Biotech, Hangzhou, China). Fluorescence of the cells was measured at an excitation wavelength of 488 nm and an emission of 530 nm using a flow cytometer (Merck kGaA, Darmstadt, Germany). The voltage of FSC, SSC and fluorescence channels were adjusted by blank tube, and under this voltage condition, the compensation of fluorescence channel was adjusted by single dye tube. After acquisition, data were exported and analyzed using FlowJo version 10. The cells were also observed under a BX51-TRF fluorescence microscope (Olympus). FITC and Rhodamine channels on this fluorescence microscope were used for observation.

Data availability
All data generated or analysed during this study are included in this published article and its supplementary information files.